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Uptake of Inorganic Mercury in the Olfactory Bulbs via Olfactory Pathways in Rats
ENVIRONMENTAL RESEARCH, SECTION A ARTICLE NO.
77, 130—140 (1998)
ER973817
Uptake of Inorganic Mercury in the Olfactory Bulbs via Olfactory Pathways in Rats1 Jo¨ rgen Henriksson and Hans Tja¨ lve Department of Pharmacology and Toxicology, Faculty of Veterinary Medicine, Swedish University of Agricultural Sciences, Box 573, SE-751 23 Uppsala, Sweden Received December 4, 1997
In humans a continuous exposure of the nasal cavity to mercury vapor (Hg0), released from amalgam fillings and oxidized to Hg21 in the olfactory mucosa, as well as a potential uptake of Hg21 in the olfactory neurons from the blood, may lead to considerable concentrations of the metal in the olfactory bulbs. ( 1998 Academic Press Key Words: amalgam; axonal transport; mercury; olfactory bulbs; olfactory neurons.
Uptake and transport in the olfactory neurons may be an important means by which some heavy metals gain access to the brain. In the present study we explored whether inorganic mercury (203Hg21) may be taken up in the CNS via the olfactory pathway. Autoradiography and gamma spectrometry showed that intranasal instillation of 203Hg21 in the right nostrils of rats resulted in much higher levels of the metal in the right olfactory bulbs than in the left ones. At the side of the application of the 203Hg21 there was also a labeling of the olfactory nerve bundles projecting to the olfactory bulbs as well as in the olfactory nerve-fibres constituting the olfactory nerve layer of the bulbs, which was not seen on the opposite side. The results also showed that the 203 Hg21 accumulated in the glomerular layer of the bulbs. These data indicate that our results can be ascribed to a movement of the mercury along the olfactory axons to their terminal parts in the glomeruli and not to circulatory uptake from the mucosal vasculature. At late survival intervals a low labeling was also discernable in the external plexiform layer, indicating that a low level of 203Hg21 leaves the terminal arborizations of the axons in the glomeruli. An uptake of 203Hg21 in the glomerular layer of the olfactory bulbs was also seen in rats given the metal intraperitoneally. This uptake was similar in the right and left bulbs and always much lower than in the right bulbs of the rats given 203 Hg21 in the right nostrils. The intraperitoneal injections in addition resulted in an uptake of the 203 Hg21 in the olfactory epithelium. We propose that in these rats the mercury is taken up from the blood into the olfactory neurons and then moves along the axons to their terminations in the olfactory bulbs.
INTRODUCTION
There is growing concern that mercury released from dental amalgam fillings may cause negative health effects (WHO, 1991; Halbach, 1994). The mercury is released from the amalgam surface mainly as mercury vapor (Hg0), which is distributed throughout the oral cavity. From there the Hg0 may be transferred via respiration to the lungs, which are considered to be the major sites of uptake of the metal. The Hg0 is partly oxidized to Hg2` in the lungs and the red blood cells, but the oxidation is considered to be slow enough to allow part of the lipophilic Hg0 to reach the brain and diffuse through the blood—brain barrier into the nervous tissue. In the brain the Hg0 can be oxidized to Hg2`, which binds to cellular nucleophiles (Sichak et al., 1986; WHO, 1991). The Hg0 released from the amalgam fillings will also reach the nasal cavity. However, little attention so far has been paid to the possible uptake of mercury from this site. The fate of the Hg0 in the nasal tissues is not known in detail, but there are indications that the Hg0 is oxidized to Hg2` in the nasal mucosa (Khayat and Dencker, 1984). We have shown that some metals, such as manganese (Mn2`), cadmium (Cd2`), and nickel (Ni2`), are taken up from the nasal olfactory epithelium to the brain via the primary olfactory neurons (Gottofrey
1
The funding of this study was supported by the Swedish Council for Work Life Research and by the Foundation for Strategic Environmental Research. All studies were conducted in accordance with national and institutional guidelines for animal welfare. 130 0013-9351/98 $25.00 Copyright ( 1998 by Academic Press All rights of reproduction in any form reserved.
UPTAKE OF INORGANIC MERCURY IN THE OLFACTORY BULBS
and Tja¨ lve, 1991; Tja¨ lve et al., 1995,1996; Henriksson et al., 1997). The cell bodies of these cells are localized in the olfactory epithelium and they have dendrites in contact with the nasal lumen and axons which reach the olfactory bulbs of the brain. The blood—brain barrier usually limits the uptake of metal cations from the systemic circulation into the brain. However, via the olfactory pathway metals may have direct access to the brain. There is a possibility that the olfactory epithelium and its neuronal connections constitute a route by which mercury released from amalgam may reach the brain. In the present study we applied inorganic mercury (203Hg2`) in the nasal cavity of rats. The uptake of the metal into the brain was then examined by autoradiography and gamma spectrometry. MATERIALS AND METHODS
Isotope HgCl2, spec. radioactivity 2.3 lCi/lg Hg2`, dissolved in 0.5 M HCl, was obtained from Amersham Sweden AB (Solna, Sweden). The isotope solution was evaporated by N2 gas and physiological saline was added to obtain a concentration of 203Hg2` of 6.7 lg (5 lCi) per 10 or 100 ll.
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1 week, and 3 weeks after i.n. instillation of 203Hg2` and 1 week and 3 weeks after i.p. administration of the metal were sectioned horizontally through the heads. Twenty-micrometer-thick sections were taken and pressed against X-ray films (Structurix D7, Agfa-Gevaert) for about 3 months. For the gamma spectrometry the levels of radioactivity were recorded in the brain, the nasal olfactory epithelium, the kidney, and the liver in rats killed 1 day and 1, 3 and 6 weeks after i.n. or i.p. administration of the 203 Hg2`. The radioactivity was determined separately in the olfactory epithelium on the left and the right side of the nasal septum. The brain parts removed included the left and right olfactory bulbs, the left and right parts of the basal areas of the cerebral hemispheres, comprising the olfactory peduncle, the olfactory tubercle, the prepiriform and periamygdaloid corticis, and the entorhinal area (these parts were designated ‘‘olfactory cortex’’), and the rest of the brain (removed in one piece).
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Statistics The Wilcoxon signed rank test was used to compare medians. Differences between groups were regarded as significant if P values were less than or equal to 0.05.
Animals Male Sprague—Dawley rats, with body weights about 150 g, were obtained from Bantin and Kingman (Sollentuna, Sweden). The animals were housed at 22°C with a 12-h light /dark cycle, with free access to tap water and a standard pellet diet (Lactamin AB, Vadstena, Sweden). Experiments Rats were given 203Hg2` (6.7 lg; 45 lg /kg body wt) either intranasally (i.n.) or intraperitoneally (i.p.). For the i.n. administration each animal was anaesthetized by pentobarbital sodium (30 mg/ kg body wt i.p.), and 10 ll (5 lCi) 203Hg2` solution was instilled into the right naris, as described previously (Tja¨ lve et al., 1996). For the i.p. injections each nonanaesthetized rat was given 100 ll (5 lCi) 203Hg2` solution. After various survival intervals the rats were killed by CO2 asphyxiation and used either for whole-body autoradiography, with tape-sections according to Ullberg et al. (1982), or for gamma spectrometry in a Packard Cobra 5003 gamma counter, which automatically corrects for isotope decay. For the autoradiography one animal each killed 1 day,
RESULTS
Autoradiography The autoradiograms of the rat killed 1 day after instillation of 203Hg2` in the right naris showed a strong labeling of the right nasal cavity and olfactory epithelium. There was a considerable labeling of the nerve bundles projecting to the right olfactory bulb. The olfactory nerve layer of the right olfactory bulb, was also labeled, and this also applied to the glomerular layer of the bulb (Fig. 1). There was no detectable radioactivity in other parts of the right bulb. The labeling was low in epithelium on the left side and in the corresponding olfactory bulb. Autoradiograms of the rat killed 1 week after instillation of 203Hg2` in the right naris showed a strong labeling of the right olfactory epithelium. There was a marked labeling of the nerve bundles projecting to the right olfactory bulb. A similar labeling was present in the nerve bundles constituting the olfactory nerve layer of this bulb. In the glomerular layer, in which the synaptic junctions are established between the axons of the primary olfactory neurons and the dendrites of the secondary olfactory neurons (the mitral and tufted cells), the radioactivity was higher than in the olfactory nerve layer of
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FIG. 1. (A) Detail of an autoradiogram of the head (horizontal section) showing the nasal cavity and the olfactory bulbs of a rat killed 1 day after instillation of 203Hg2` (6.7 lg) in the right nostril. (B) The corresponding tissue section. epl, external plexiform layer of the right olfactory bulb; gl, glomerular layer of the right olfactory bulb; lb, left olfactory bulb; ml, mitralis cell layer of the right olfactory bulb; nb, nerve bundles from the right olfactory epithelium; onl, olfactory nerve layer of the right olfactory bulb; rb, right olfactory bulb; re, right olfactory epithelium. Magnification, ]16.
the bulb (Fig. 2). There was a labeling of the external plexiform layer which was much weaker than in the glomerular layer. This labeling was strongest adjacent to the glomerular layer and faded out toward the mitralis cell layer. The labeling was low in the epithelium on the left side of the nasal septum and in the left olfactory bulb (Fig. 2). The autoradiograms of the rat killed 3 weeks after instillation of 203Hg2` in the right naris showed a weaker labeling of the various structures in the nasal cavity and the olfactory bulb than at the previous survival interval. In the right olfactory bulb the radioactivity in the nerve layer was very low,
whereas the labeling of the glomerular layer was distinct (Fig. 3). There was also a labeling of the external plexiform layer, which was lower than in the glomerular layer and fading out toward the mitral cell layer. A very low radioactivity was present in the right olfactory epithelium and in the nerve bundles present in the submucosa and penetrating the ethmoid bone. In the autoradiograms of the rat killed 1 week after i.p. administration of the 203Hg2` there was a considerable labeling of the olfactory epithelium on both sides of the nasal septum. In the right and left olfactory bulbs there was a considerable labeling of
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FIG. 2. (A) Detail of an autoradiogram of the head (horizontal section) showing the nasal cavity and the olfactory bulbs of a rat killed 1 week after instillation of 203Hg2` (6.7 lg) in the right nostril. (B) The corresponding tissue section. epl, external plexiform layer of the right olfactory bulb; gl, glomerular layer of the right olfactory bulb; lb, left olfactory bulb; ml, mitralis cell layer of the right olfactory bulb; nb, nerve bundles from the right olfactory epithelium; onl, olfactory nerve layer of the right olfactory bulb; rb, right olfactory bulb; re, right olfactory epithelium. Magnification, ]16.
the glomerular layer. Other parts of the olfactory bulbs showed a low radioactivity (Fig. 4). The radioactivity in most areas of the brain outside the olfactory bulbs showed a labeling similar to the weakly labeled areas of the latter. However, the radioactivity in some areas of the di- and mesencephalon, such as the habenula and the superior and inferior colliculi (most marked in the latter), slightly exceeded the ‘‘background labeling’’ present in most parts of the brain. There was a marked labelling of the plexus chor-
ioideus. The optic nerves, which were visible at their exit from the orbits, showed a considerable radioactivity (Fig. 4). In the eye there was a weak labeling of the sclera and an even weaker of the retina. In the autoradiograms of the rat killed 3 weeks after i.p. injection of 203Hg2` a low labeling was seen in the glomerular layer of the olfactory bulbs (Fig. 5). The labeling of other parts of the brain was hardly discernable. A weak labeling was also present in the olfactory epithelium.
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FIG. 3. (A) Detail of an autoradiogram of the head (horizontal section) showing the nasal cavity and the olfactory bulbs of a rat killed 3 weeks after instillation of 203Hg2` (6.7 lg) in the right nostril. (B) The corresponding tissue section. epl, external plexiform layer of the right olfactory bulb; gl, glomerular layer of the right olfactory bulb; lb, left olfactory bulb; ml, mitralis cell layer of the right olfactory bulb; onl, olfactory nerve layer of the right olfactory bulb; rb, right olfactory bulb; re, right olfactory epithelium. Magnification, ]16.
Gamma Spectrometry Intranasal instillation of 203Hg2`. Table 1 shows that the uptake of 203Hg2` in the right olfactory bulb
is significantly higher than in all other areas of the brain at all survival intervals. The levels of the metal in this bulb are somewhat lower at 1 week than at 1 day. At 3 weeks the concentration has
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FIG. 4. (A) Detail of an autoradiogram of the head (horizontal section) showing the nasal cavity and the olfactory bulbs of a rat killed 1 w after i.p. injection of 203Hg2` (6.7 lg). (B) The corresponding tissue section. e, olfactory epithelium; h, habenula; gl, glomerular layer of the olfactory bulbs; ic, inferior colliculus; on, optic nerve; pc, plexus chorioideus; r, retina; s, sclera; sc, superior colliculus. Original magnification, ]8.
decreased to about one-fourth of the value of 1 week and at 6 weeks there is a further reduction to about half of the concentration observed at 3 weeks. The concentration of 203Hg2` in the left olfactory bulb is much lower than in the right bulb, but still higher than in the other parts of the brain. It is known that
the nasal septum in rats contains the so-called ‘‘septal window’’ (Kelemen and Sargent, 1946). This implies that a part of the metal applied in the right nostril may pass over to the left side. In the right and left olfactory cortices there are similar levels of the metal at the respective survival intervals and these
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FIG. 5. (A) Detail of an autoradiogram of the head (horizontal section) showing the nasal cavity and the olfactory bulbs of a rat killed 3 weeks after i.p. injection of 203Hg2` (6.7 lg). (B) The corresponding tissue section. e, olfactory epithelium; gl, glomerular layer of the olfactory bulbs. Magnification, ]16.
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TABLE 1
TABLE 2
Concentration of 203Hg21 in Some Neuronal and Extraneuronal Tissues of Rats at Different Intervals after Instillation of the Metal in the Right Nostril (45 lg/kg body wt)
Concentration of 203Hg21 in Some Neuronal and Extraneuronal Tissues of Rats at Different Intervals after Intraperitoneal Administration of the Metal (45 lg/kg body wt) Tissue-concentration of 203Hg2` (ng/g of wet weight)a
Tissue concentration of 203Hg2# (ng/g of wet weight)a 1 day Olfactory epithelium Olfactory bulb Olfactory cortex Brainc Liver Kidney
1 week
R 5801$1494
128$14
L R L R L
22$5.9 80$21 6.1$2.5b 1.6$0.3b 1.3$0.2b 1.5$0.1b 20$3.0 274$38
62$18 109$27 9.7$7.4b 1.9$1.0b 2.5$0.6b 1.3$0.2b 35$5.5 235$19
3 weeks
6 weeks
18$4.1
8.4$2.6
4.0$1.2 23$14 2.0$0.8b 0.3$0.1b 0.2$0.1b 0.5$0.1b 2.9$0.8 138$25
1.4$0.8 9.4$1.7 1.6$1.3b 0.3$0.2b 0.3$0.2b 0.2$0.1b 0.7$0.1 99$23
Note. R, right; L, left. a means$SD, n"5—6 rats. b P40.05 compared to the right olfactory bulb (Wilcoxon signed rank test). c Brain except olfactory bulb and olfactory cortex.
levels are also similar to the ones observed in the rest of the brain. The concentrations of 203Hg2` in the right olfactory epithelium decrease from 1 day to 1 week (Table 1). At 3 and 6 weeks the levels in the right olfactory epithelium are similar to those in the right olfactory bulb. The concentrations of the metal in the left olfactory epithelium are much lower than in the right epithelium at all intervals. The data in Table 1 also show that the kidneys accumulate considerable levels of 203Hg2` after intranasal administration of the metal. This concentration is retained at a high level also after 6 weeks. The concentration of 203Hg2` in the liver is much lower than in the kidney and also lower than in the right olfactory bulb at all intervals. Intraperitoneal administration of 203Hg2`. After i.p. administration of 203Hg2` the concentrations of the metal in the right and left olfactory epithelium, in the right and left olfactory bulbs, and in the right and left olfactory cortices, respectively, showed similar values at the various survival intervals. Therefore, for the calculation of the results in Table 2 no dividing in right and left was done. It can be seen in Table 2 that the concentrations of 203Hg2` in the olfactory epithelium are considerable higher than in the brain at 1 day and 1 week. In the brain of these rats the 203Hg2` concentrations in the olfactory bulbs are statistically higher than in the olfactory cortex and the rest of the brain at all survival intervals.
Olfactory epithelium Olfactory bulbs Olfactory cortex Brainb Liver Kidney a b c
1 day
1 week
3 weeks
6 weeks
22$1.2
12$1.4
3.2$0.3
0.9$0.1
2.5$0.3 2.2$0.1b 1.8$0.3b 73$7.4 895$65
3.9$0.3 2.6$0.3b 2.9$0.2b 39$9.0 832$71
2.3$0.2 1.0$0.3b 1.3$0.1b 6.4$1.3 445$46
0.9$0.1 0.3$0.1b 0.4$0.1b 1.6$0.4 274$50
means$SD, n"5—6 rats. P40.05 compared to the olfactory bulbs (Wilcoxon signed rank test). Brain except olfactory bulb and olfactory cortex.
However, a comparison of the data in Table 2 with those in Table 1 shows that the high labeling of the right olfactory bulb is unique for the rats given the 203 Hg2` in the right nostril. At the 1- and 3-week survival intervals the levels of 203Hg2` in the olfactory cortex and the rest of the brain appear to be somewhat higher in the i.p. injected rats than in the i.n. injected ones, whereas at the 1-day and 6-week survival intervals similar values are seen in the i.p. and i.n. injected rats. A comparison of the data in Table 2 and Table 1 further shows that the concentrations of 203Hg2` in the kidney are higher after i.p. administration of the metal than after i.n. instillation at the corresponding survival intervals. Also the liver contains higher concentrations of the metal after i.p. compared to i.n. administration of the metal. Right—left ratios of 203Hg2`. It can be seen in Fig. 6 that in the rats given the metal in the right nostril the ratios between the right and left olfactory epithelium are higher than in the right and left olfactory bulb at 1 day. It can also be noted that the two rats which have the lowest ratios in the olfactory epithelium also have the lowest ratios in the olfactory bulbs. At the 1-, 3-, and 6-week survival intervals the ratios between the right and left olfactory epithelium and the right and left olfactory bulbs show similar values. In the olfactory cortex the ratios are close to 1 at all intervals after i.n. instillation of the metal. In the rats given 203Hg2` i.p. the ratios between the levels of the metal on the right and left side are close to 1 in the olfactory epithelium, the olfactory bulb, and the olfactory cortex at all survival intervals.
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FIG. 6. Ratios between the concentrations of 203Hg2` in the olfactory epithelium on the right and left side of the nasal septum and in the olfactory bulb and the olfactory cortex on the right and left side of the midline of the brain in rats given the metal i.n. or i.p. The rats were killed at different intervals after administration of the 203Hg2` (6.7 lg). The intranasal instillations were done in the right nostril. Each curve shows the data obtained in a single rat.
DISCUSSION
The demonstration that instillation of 203Hg2` in the right nostrils of the rats resulted in much higher levels of the metal in the right olfactory bulbs than in the left ones, together with the observation of absence of a similar uptake after i.p. administration, support the assumption that there is a direct passage of the metal from the olfactory epithelium to the olfactory bulbs. At the side of the application of the 203 Hg2` there was a labeling of the olfactory nerve bundles projecting to the olfactory bulbs, as well as in the olfactory neve-fibres constituting the olfactory nerve layer of the olfactory bulbs, which was not seen on the opposite side. The results also showed that the 203Hg2` accumulated in the glomerular layer of the bulbs. These data indicate that our observations can be ascribed to a movement of the mercury along the olfactory axons to their terminal parts in the glomeruli and not to circulatory uptake from the mucosal vasculature. The labeling was more selectively localized to the glomerular layer at 3 weeks than at 1 week. At 3 weeks there is a low source of mercury available for transport from the olfactory epithelium and most of the 203Hg2` appears
to have reached the axonal terminations in the glomeruli. At the 1- and 3-week survival intervals a weak labeling was also discernable in the external plexiform layer. This observation indicates that a low level of 203Hg2` leaves the terminal arborizations of the axons in the glomeruli. However, the labeling of the olfactory cortex, i.e., the projection field of the secondary olfactory neurons, did not differ from other ‘‘extrabulbar’’ brain areas. This indicates that the mercury which leaves the terminal parts of the primary olfactory neurons is not transferred via secondary olfactory neurons. Rather the mercury may diffuse in the extracellular space of the external plexiform layer. That there is a lack of transport of mercury via secondary olfactory neurons is further substantiated by the observation that the levels of the metal were similar in the right and left olfactory cortices of the rats given the 203Hg2` intranasally. In addition the levels of mercury in the olfactory cortex of the rats given the 203Hg2` i.p. did not differ very much from the levels in the i.n. injected rats and these concentrations were also similar to those observed in the rest of the brain. These data indicate that the mercury in the olfactory cortex and in the other extrabulbar areas is taken up from the blood.
UPTAKE OF INORGANIC MERCURY IN THE OLFACTORY BULBS
We have previously shown that 203Hg2` is taken up from the olfactory epithelium along the olfactory neurons to the olfactory bulbs of pikes (Borg-Neczak and Tja¨ lve, 1996). This transport occurs in an anterograde direction. Studies in rodents by Arvidson (1992) and Schi+nning (1993) have shown that inorganic mercury (Hg2`) is taken up in nerve terminals in skeletal muscles and then transported retrogradely along the motoneurons to the cell bodies in the spinal cord, the brainstem, and the dorsal root ganglia. Schi+nning (1993) reported that intraneuronal injection of colchicine, which is an inhibitor of cellular functions dependent on microtubules, suppressed the retrograde transport of mercury in the rat sciatic nerve, indicating an active intraneuronal movement of the metal. Further studies are required to determine the mechanism for the uptake of the mercury in the olfactory neurons. Following intranasal administration the mercury is probably taken up in the olfactory knobs, which constitute the terminal swellings of the dendritic processes, extending to the surface of the epithelium. It is known that the olfactory knobs are rich in endocytic vesicles, indicating that they are actively engaged in endocytosis of exogeneous materials from the extracellular environment (de Lorenzo, 1970; Bannister and Dodson, 1992). Entry of mercury by endocytotis may involve a binding of the metal to surface proteins followed by internalization. It is known that such a mechanism is involved in the uptake of various macromolecules in nerve terminals (Yamamoto et al., 1987). In addition Gutknecht (1981) suggested that Hg2` may pass biological membranes as an uncharged HgCl2 complex. The Hg2` may also enter the cells via sodium or calcium channels, as has been proposed as the mode of uptake of Hg2` in motor nerve terminals in frog muscle (Miyamoto, 1983). The autoradiography showed an uptake of 203Hg2` in the glomerular layer of the olfactory bulbs also after i.p. administration of the metal. This observation was supported by the gamma spectrometry, which showed significantly higher levels of mercury in the olfactory bulbs than in other parts of the brain. It can be noted that the gamma spectrometry reflects the average mercury concentration in the bulbs and not specificially the uptake in the glomerular layer. The results also showed an uptake of the mercury in the olfactory epithelium in the i.p. injected rats. We assume that the mercury is taken up from the blood into the primary olfactory neurons and then moves along the axons to their terminations in the bulb. Results by Arvidson (1992) have indicated a movement of mercury from peripheral
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nerve terminals along motoneurons to their cell bodies in the spinal cord and brainstem following intravenous injection of Hg2` in mice. In the rats injected i.p. with 203Hg2` we observed a labeling of the superior and inferior colliculi which slightly exceeded the background labeling of the brain. We also found a labeling of the optic nerves. The superior colliculus is a terminal of the axons of the retinal ganglion cells and conceivably the mercury may reach this area via transport in the optic nerves. The inferior colliculus is a terminal of the central auditory tract. Conceivably the mercury may reach this area via neurons in the auditory system. The habenula also showed a somewhat higher labeling than the background in the brain. The habenula receives fibers from the olfactory cortex via the olfactory—habenular tract (Allison, 1953), but it is not currently known whether mercury can be transported along this route. Our results showed a high accumulation of 203 Hg2` in plexus chorioideus in the rats given the metal i.p. It is known that inorganic mercury is accumulated in this structure (Berlin and Ullberg, 1963). As mentioned in the Introduction, there are indications that Hg0 is oxidized to Hg2` in the nasal mucosa (Khayat and Dencker, 1984). The oxidation is carried out by the H2O2-catalase pathway, which is ubiquitously present in various tissues (Sichak et al., 1986; Clarkson, 1989). It is well known that Hg0 is oxidized to Hg2` in the CNS. We propose that Hg0 released from amalgam fillings may reach the primary olfactory neurons in the olfactory epithelium and become oxidized to Hg2` in these cells. The intracellular Hg2` may then be transported along the axons. In humans a continuous exposure of the nasal mucosa to mercury released from amalgam fillings, as well as a potential uptake of the metal in the olfactory epithelium from the blood, may result in considerable concentrations of the metal in the olfactory bulbs. Maas and co-workers (1996) examined the concentration of mercury in the olfactory bulbs of 55 deceased persons and found significantly higher levels of the metal in the bulbs than in the occipital lobe cortex (geometric mean 17.4 lg/kg wet wt compared to 9.2 lg/kg wet wt). However, the levels of mercury in the olfactory bulbs could not be correlated to the number of amalgam fillings. It would be of interest to further explore the concentration of mercury in the olfactory bulbs in humans. The potential toxicity of mercury for the olfactory sense is also an issue which needs to be explored. To our knowledge this problem has not been examined
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in mammals. However, the olfactory system has been shown to be very vulnerable to mercury in fish (Hara et al., 1976; Baatrup et al., 1990). As mentioned Hg2` has been shown to be transported via the olfactory pathway in pike (Borg-Neczak and Tja¨ lve, 1996). It is also of interest to notice that the olfactory system has been shown to be impared in early stages of some neurodegenerative disorders, such as Alzheimer’s disease and Parkinson’s disease, and it has been proposed that an uptake of neurotoxic materials along the olfactory pathways may contribute to these diseases (Roberts, 1986; Ferrera-Moyano and Barragan, 1989; Doty, 1991). Therefore, toxicants which are able to enter the brain via the olfactory system deserve special attention. REFERENCES Allison, A. C. (1953). The morphology of the olfactory system in the vertebrates. Biol. Rev. 28, 195—244. Arvidson, B. (1992). Inorganic mercury is transported from muscular nerve terminals to spinal and brainstem motoneurons. Muscle Nerve 15, 1089—1094. Baatrup, E., D+ving, K. B., and Winberg, S. (1990). Differential effects of mercurial compounds on the electroolfactogram (EOG) of salmon (Salmo salar L.) Ecotoxicol. Environ. Saf. 20, 269—276. Berlin, M., and Ullberg, S. (1963). Accumulation and retention of mercury in the mouse. Arch. Environ. Health 6, 589—601. Bannister, L. H., and Dodson, H. C. (1992). Endocytic pathways in the olfactory and vomeronasal epithelia of the mouse: Ultrastructure and uptake of tracers. Microsc. Res. Tech. 23, 128—141. Borg-Neczak, K., and Tja¨ lve, H. (1996). Uptake of 203Hg2` in the olfactory system in pike. Toxicol. Lett. 84, 107—112. Clarkson, T. W. (1989). Mercury. J. Am. Coll. Toxicol. 8, 12911295. de Lorenzo, A. J. D. (1970). The olfactory neuron and the blood—brain barrier. In ‘‘Taste and Smell in Vertebrates’’ (G. E. W. Wolstenholme and J. Knight, Eds.), pp. 151—176. Churchill, London. Doty, R. L. (1991). Olfactory dysfunction in neurodegenerative disorders. In ‘‘Smell and Taste in Health and Disease’’ (T. V. Getchell, L. M. Bartoshuk, R. L. Doty, and J. B. Snow, Jr., Eds.), pp. 735—751. Raven Press, New York. Ferrera-Moyano, H., and Barragan, E. (1989). The olfactory system and Alzheimer’s disease. Int. J. Neurosci. 49, 157—197.
Gottofrey, J., and Tja¨ lve, H. (1991). Axonal transport of cadmium in the olfactory nerve of the pike. Pharmacol. Toxicol. 69, 242—252. Gutknecht, J. (1981). Inorganic mercury (Hg2`) transport through lipid bilayer membranes. J. Membr. Biol. 61, 61-66. Halbach, S. (1994). Amalgam tooth fillings and man’s mercury burden. Hum. Exp. Toxicol. 13, 496-501. Hara, T. J., Law, Y. M. C., and Macdonald, S. (1976). Effects of mercury and copper on the olfactory response in rainbow trout, Salmo gairdneri. J. Fish. Res. Board Can. 33, 1568—1573. Henriksson, J., Tallkvist, J., and Tja¨ lve, H. (1997). Uptake of nickel into the brain via olfactory neurons in rats. Toxicol. Lett. 91, 153—162. Kelemen, G., and Sargent, F. (1946). Nonexperimental pathological nasal findings in laboratory rats. Arch. Otolaryngol. 44, 24-42. Khayat, A., and Dencker, L. (1984). Organ and cellular distribution of inhaled metallic mercury in the rat and Marmoset monkey (Callithrix jacchus): Influence of ethyl alcohol pretreatment. Acta. Pharmacol. Toxicol. 55, 145—152. Maas, C., Bru¨ ck, H., Haffner, H.-T., and Schweinsberg, F. (1996). Investigations on cerebral mercury from dental amalgam fillings through a direct nose-brain transport. Zentralbl. Hyg. 198, 275—291. Miyamoto, M. T. (1983). Hg2` causes neurotoxicity at an intracellular site following entry through Na and Ca channels. Brain. Res. 267, 375—379. Roberts, E. (1986). Alzheimer’s disease may begin in the nose and may be caused by alumino silicates. Neurobiol. Aging 7, 561—567. Schi+nning, J. D. (1993). Retrograde axonal transport of mercury in rat sciatic nerve. Toxicol. Appl. Pharmacol. 121, 43—49. Sichak, S. P., Mavis, R. D., Finkelstein, J. N., and Clarkson, T. W. (1986). An examination of the oxidation of mercury vapor by rat brain homogenate. J. Biochem. Toxicol. 1, 53—68. Tja¨ lve, H., Meja` re, C., and Borg-Neczak, K. (1995). Uptake and transport of manganese in primary and secondary olfactory neurons in pike. Pharmacol. Toxicol. 77, 23—31. Tja¨ lve, H., Henriksson, J., Tallkvist, J., Larsson, B. S., and Lindquist, N. G. (1996). Uptake of manganese and cadmium from the nasal mucosa into the central nervous system via olfactory pathways in rats. Pharmacol. Toxicol. 79, 347—356. Ullberg, S., Larsson, B., and Tja¨ lve, H. (1982). Autoradiography. In ‘‘Biological Applications of Radiotracers’’ (H. J. Glenn and L. G. Colombetti, Eds.), pp. 55-108. CRC Press, Boca Raton. WHO (1991). ‘‘Inorganic Mercury.’’ Environmental Health Criteria 118, World Health Organization, Geneva. Yamamoto, T., Iwasaki, Y., Kouno, H., Iizuka, H., and Zhao, J.-X. (1987). Retrograde transport and differential accumulation of serum proteins in motor neurons. Implication for motor neuron diseases. Neurology 37, 843—846.
77, 130—140 (1998)
ER973817
Uptake of Inorganic Mercury in the Olfactory Bulbs via Olfactory Pathways in Rats1 Jo¨ rgen Henriksson and Hans Tja¨ lve Department of Pharmacology and Toxicology, Faculty of Veterinary Medicine, Swedish University of Agricultural Sciences, Box 573, SE-751 23 Uppsala, Sweden Received December 4, 1997
In humans a continuous exposure of the nasal cavity to mercury vapor (Hg0), released from amalgam fillings and oxidized to Hg21 in the olfactory mucosa, as well as a potential uptake of Hg21 in the olfactory neurons from the blood, may lead to considerable concentrations of the metal in the olfactory bulbs. ( 1998 Academic Press Key Words: amalgam; axonal transport; mercury; olfactory bulbs; olfactory neurons.
Uptake and transport in the olfactory neurons may be an important means by which some heavy metals gain access to the brain. In the present study we explored whether inorganic mercury (203Hg21) may be taken up in the CNS via the olfactory pathway. Autoradiography and gamma spectrometry showed that intranasal instillation of 203Hg21 in the right nostrils of rats resulted in much higher levels of the metal in the right olfactory bulbs than in the left ones. At the side of the application of the 203Hg21 there was also a labeling of the olfactory nerve bundles projecting to the olfactory bulbs as well as in the olfactory nerve-fibres constituting the olfactory nerve layer of the bulbs, which was not seen on the opposite side. The results also showed that the 203 Hg21 accumulated in the glomerular layer of the bulbs. These data indicate that our results can be ascribed to a movement of the mercury along the olfactory axons to their terminal parts in the glomeruli and not to circulatory uptake from the mucosal vasculature. At late survival intervals a low labeling was also discernable in the external plexiform layer, indicating that a low level of 203Hg21 leaves the terminal arborizations of the axons in the glomeruli. An uptake of 203Hg21 in the glomerular layer of the olfactory bulbs was also seen in rats given the metal intraperitoneally. This uptake was similar in the right and left bulbs and always much lower than in the right bulbs of the rats given 203 Hg21 in the right nostrils. The intraperitoneal injections in addition resulted in an uptake of the 203 Hg21 in the olfactory epithelium. We propose that in these rats the mercury is taken up from the blood into the olfactory neurons and then moves along the axons to their terminations in the olfactory bulbs.
INTRODUCTION
There is growing concern that mercury released from dental amalgam fillings may cause negative health effects (WHO, 1991; Halbach, 1994). The mercury is released from the amalgam surface mainly as mercury vapor (Hg0), which is distributed throughout the oral cavity. From there the Hg0 may be transferred via respiration to the lungs, which are considered to be the major sites of uptake of the metal. The Hg0 is partly oxidized to Hg2` in the lungs and the red blood cells, but the oxidation is considered to be slow enough to allow part of the lipophilic Hg0 to reach the brain and diffuse through the blood—brain barrier into the nervous tissue. In the brain the Hg0 can be oxidized to Hg2`, which binds to cellular nucleophiles (Sichak et al., 1986; WHO, 1991). The Hg0 released from the amalgam fillings will also reach the nasal cavity. However, little attention so far has been paid to the possible uptake of mercury from this site. The fate of the Hg0 in the nasal tissues is not known in detail, but there are indications that the Hg0 is oxidized to Hg2` in the nasal mucosa (Khayat and Dencker, 1984). We have shown that some metals, such as manganese (Mn2`), cadmium (Cd2`), and nickel (Ni2`), are taken up from the nasal olfactory epithelium to the brain via the primary olfactory neurons (Gottofrey
1
The funding of this study was supported by the Swedish Council for Work Life Research and by the Foundation for Strategic Environmental Research. All studies were conducted in accordance with national and institutional guidelines for animal welfare. 130 0013-9351/98 $25.00 Copyright ( 1998 by Academic Press All rights of reproduction in any form reserved.
UPTAKE OF INORGANIC MERCURY IN THE OLFACTORY BULBS
and Tja¨ lve, 1991; Tja¨ lve et al., 1995,1996; Henriksson et al., 1997). The cell bodies of these cells are localized in the olfactory epithelium and they have dendrites in contact with the nasal lumen and axons which reach the olfactory bulbs of the brain. The blood—brain barrier usually limits the uptake of metal cations from the systemic circulation into the brain. However, via the olfactory pathway metals may have direct access to the brain. There is a possibility that the olfactory epithelium and its neuronal connections constitute a route by which mercury released from amalgam may reach the brain. In the present study we applied inorganic mercury (203Hg2`) in the nasal cavity of rats. The uptake of the metal into the brain was then examined by autoradiography and gamma spectrometry. MATERIALS AND METHODS
Isotope HgCl2, spec. radioactivity 2.3 lCi/lg Hg2`, dissolved in 0.5 M HCl, was obtained from Amersham Sweden AB (Solna, Sweden). The isotope solution was evaporated by N2 gas and physiological saline was added to obtain a concentration of 203Hg2` of 6.7 lg (5 lCi) per 10 or 100 ll.
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1 week, and 3 weeks after i.n. instillation of 203Hg2` and 1 week and 3 weeks after i.p. administration of the metal were sectioned horizontally through the heads. Twenty-micrometer-thick sections were taken and pressed against X-ray films (Structurix D7, Agfa-Gevaert) for about 3 months. For the gamma spectrometry the levels of radioactivity were recorded in the brain, the nasal olfactory epithelium, the kidney, and the liver in rats killed 1 day and 1, 3 and 6 weeks after i.n. or i.p. administration of the 203 Hg2`. The radioactivity was determined separately in the olfactory epithelium on the left and the right side of the nasal septum. The brain parts removed included the left and right olfactory bulbs, the left and right parts of the basal areas of the cerebral hemispheres, comprising the olfactory peduncle, the olfactory tubercle, the prepiriform and periamygdaloid corticis, and the entorhinal area (these parts were designated ‘‘olfactory cortex’’), and the rest of the brain (removed in one piece).
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Statistics The Wilcoxon signed rank test was used to compare medians. Differences between groups were regarded as significant if P values were less than or equal to 0.05.
Animals Male Sprague—Dawley rats, with body weights about 150 g, were obtained from Bantin and Kingman (Sollentuna, Sweden). The animals were housed at 22°C with a 12-h light /dark cycle, with free access to tap water and a standard pellet diet (Lactamin AB, Vadstena, Sweden). Experiments Rats were given 203Hg2` (6.7 lg; 45 lg /kg body wt) either intranasally (i.n.) or intraperitoneally (i.p.). For the i.n. administration each animal was anaesthetized by pentobarbital sodium (30 mg/ kg body wt i.p.), and 10 ll (5 lCi) 203Hg2` solution was instilled into the right naris, as described previously (Tja¨ lve et al., 1996). For the i.p. injections each nonanaesthetized rat was given 100 ll (5 lCi) 203Hg2` solution. After various survival intervals the rats were killed by CO2 asphyxiation and used either for whole-body autoradiography, with tape-sections according to Ullberg et al. (1982), or for gamma spectrometry in a Packard Cobra 5003 gamma counter, which automatically corrects for isotope decay. For the autoradiography one animal each killed 1 day,
RESULTS
Autoradiography The autoradiograms of the rat killed 1 day after instillation of 203Hg2` in the right naris showed a strong labeling of the right nasal cavity and olfactory epithelium. There was a considerable labeling of the nerve bundles projecting to the right olfactory bulb. The olfactory nerve layer of the right olfactory bulb, was also labeled, and this also applied to the glomerular layer of the bulb (Fig. 1). There was no detectable radioactivity in other parts of the right bulb. The labeling was low in epithelium on the left side and in the corresponding olfactory bulb. Autoradiograms of the rat killed 1 week after instillation of 203Hg2` in the right naris showed a strong labeling of the right olfactory epithelium. There was a marked labeling of the nerve bundles projecting to the right olfactory bulb. A similar labeling was present in the nerve bundles constituting the olfactory nerve layer of this bulb. In the glomerular layer, in which the synaptic junctions are established between the axons of the primary olfactory neurons and the dendrites of the secondary olfactory neurons (the mitral and tufted cells), the radioactivity was higher than in the olfactory nerve layer of
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FIG. 1. (A) Detail of an autoradiogram of the head (horizontal section) showing the nasal cavity and the olfactory bulbs of a rat killed 1 day after instillation of 203Hg2` (6.7 lg) in the right nostril. (B) The corresponding tissue section. epl, external plexiform layer of the right olfactory bulb; gl, glomerular layer of the right olfactory bulb; lb, left olfactory bulb; ml, mitralis cell layer of the right olfactory bulb; nb, nerve bundles from the right olfactory epithelium; onl, olfactory nerve layer of the right olfactory bulb; rb, right olfactory bulb; re, right olfactory epithelium. Magnification, ]16.
the bulb (Fig. 2). There was a labeling of the external plexiform layer which was much weaker than in the glomerular layer. This labeling was strongest adjacent to the glomerular layer and faded out toward the mitralis cell layer. The labeling was low in the epithelium on the left side of the nasal septum and in the left olfactory bulb (Fig. 2). The autoradiograms of the rat killed 3 weeks after instillation of 203Hg2` in the right naris showed a weaker labeling of the various structures in the nasal cavity and the olfactory bulb than at the previous survival interval. In the right olfactory bulb the radioactivity in the nerve layer was very low,
whereas the labeling of the glomerular layer was distinct (Fig. 3). There was also a labeling of the external plexiform layer, which was lower than in the glomerular layer and fading out toward the mitral cell layer. A very low radioactivity was present in the right olfactory epithelium and in the nerve bundles present in the submucosa and penetrating the ethmoid bone. In the autoradiograms of the rat killed 1 week after i.p. administration of the 203Hg2` there was a considerable labeling of the olfactory epithelium on both sides of the nasal septum. In the right and left olfactory bulbs there was a considerable labeling of
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FIG. 2. (A) Detail of an autoradiogram of the head (horizontal section) showing the nasal cavity and the olfactory bulbs of a rat killed 1 week after instillation of 203Hg2` (6.7 lg) in the right nostril. (B) The corresponding tissue section. epl, external plexiform layer of the right olfactory bulb; gl, glomerular layer of the right olfactory bulb; lb, left olfactory bulb; ml, mitralis cell layer of the right olfactory bulb; nb, nerve bundles from the right olfactory epithelium; onl, olfactory nerve layer of the right olfactory bulb; rb, right olfactory bulb; re, right olfactory epithelium. Magnification, ]16.
the glomerular layer. Other parts of the olfactory bulbs showed a low radioactivity (Fig. 4). The radioactivity in most areas of the brain outside the olfactory bulbs showed a labeling similar to the weakly labeled areas of the latter. However, the radioactivity in some areas of the di- and mesencephalon, such as the habenula and the superior and inferior colliculi (most marked in the latter), slightly exceeded the ‘‘background labeling’’ present in most parts of the brain. There was a marked labelling of the plexus chor-
ioideus. The optic nerves, which were visible at their exit from the orbits, showed a considerable radioactivity (Fig. 4). In the eye there was a weak labeling of the sclera and an even weaker of the retina. In the autoradiograms of the rat killed 3 weeks after i.p. injection of 203Hg2` a low labeling was seen in the glomerular layer of the olfactory bulbs (Fig. 5). The labeling of other parts of the brain was hardly discernable. A weak labeling was also present in the olfactory epithelium.
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FIG. 3. (A) Detail of an autoradiogram of the head (horizontal section) showing the nasal cavity and the olfactory bulbs of a rat killed 3 weeks after instillation of 203Hg2` (6.7 lg) in the right nostril. (B) The corresponding tissue section. epl, external plexiform layer of the right olfactory bulb; gl, glomerular layer of the right olfactory bulb; lb, left olfactory bulb; ml, mitralis cell layer of the right olfactory bulb; onl, olfactory nerve layer of the right olfactory bulb; rb, right olfactory bulb; re, right olfactory epithelium. Magnification, ]16.
Gamma Spectrometry Intranasal instillation of 203Hg2`. Table 1 shows that the uptake of 203Hg2` in the right olfactory bulb
is significantly higher than in all other areas of the brain at all survival intervals. The levels of the metal in this bulb are somewhat lower at 1 week than at 1 day. At 3 weeks the concentration has
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FIG. 4. (A) Detail of an autoradiogram of the head (horizontal section) showing the nasal cavity and the olfactory bulbs of a rat killed 1 w after i.p. injection of 203Hg2` (6.7 lg). (B) The corresponding tissue section. e, olfactory epithelium; h, habenula; gl, glomerular layer of the olfactory bulbs; ic, inferior colliculus; on, optic nerve; pc, plexus chorioideus; r, retina; s, sclera; sc, superior colliculus. Original magnification, ]8.
decreased to about one-fourth of the value of 1 week and at 6 weeks there is a further reduction to about half of the concentration observed at 3 weeks. The concentration of 203Hg2` in the left olfactory bulb is much lower than in the right bulb, but still higher than in the other parts of the brain. It is known that
the nasal septum in rats contains the so-called ‘‘septal window’’ (Kelemen and Sargent, 1946). This implies that a part of the metal applied in the right nostril may pass over to the left side. In the right and left olfactory cortices there are similar levels of the metal at the respective survival intervals and these
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FIG. 5. (A) Detail of an autoradiogram of the head (horizontal section) showing the nasal cavity and the olfactory bulbs of a rat killed 3 weeks after i.p. injection of 203Hg2` (6.7 lg). (B) The corresponding tissue section. e, olfactory epithelium; gl, glomerular layer of the olfactory bulbs. Magnification, ]16.
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TABLE 1
TABLE 2
Concentration of 203Hg21 in Some Neuronal and Extraneuronal Tissues of Rats at Different Intervals after Instillation of the Metal in the Right Nostril (45 lg/kg body wt)
Concentration of 203Hg21 in Some Neuronal and Extraneuronal Tissues of Rats at Different Intervals after Intraperitoneal Administration of the Metal (45 lg/kg body wt) Tissue-concentration of 203Hg2` (ng/g of wet weight)a
Tissue concentration of 203Hg2# (ng/g of wet weight)a 1 day Olfactory epithelium Olfactory bulb Olfactory cortex Brainc Liver Kidney
1 week
R 5801$1494
128$14
L R L R L
22$5.9 80$21 6.1$2.5b 1.6$0.3b 1.3$0.2b 1.5$0.1b 20$3.0 274$38
62$18 109$27 9.7$7.4b 1.9$1.0b 2.5$0.6b 1.3$0.2b 35$5.5 235$19
3 weeks
6 weeks
18$4.1
8.4$2.6
4.0$1.2 23$14 2.0$0.8b 0.3$0.1b 0.2$0.1b 0.5$0.1b 2.9$0.8 138$25
1.4$0.8 9.4$1.7 1.6$1.3b 0.3$0.2b 0.3$0.2b 0.2$0.1b 0.7$0.1 99$23
Note. R, right; L, left. a means$SD, n"5—6 rats. b P40.05 compared to the right olfactory bulb (Wilcoxon signed rank test). c Brain except olfactory bulb and olfactory cortex.
levels are also similar to the ones observed in the rest of the brain. The concentrations of 203Hg2` in the right olfactory epithelium decrease from 1 day to 1 week (Table 1). At 3 and 6 weeks the levels in the right olfactory epithelium are similar to those in the right olfactory bulb. The concentrations of the metal in the left olfactory epithelium are much lower than in the right epithelium at all intervals. The data in Table 1 also show that the kidneys accumulate considerable levels of 203Hg2` after intranasal administration of the metal. This concentration is retained at a high level also after 6 weeks. The concentration of 203Hg2` in the liver is much lower than in the kidney and also lower than in the right olfactory bulb at all intervals. Intraperitoneal administration of 203Hg2`. After i.p. administration of 203Hg2` the concentrations of the metal in the right and left olfactory epithelium, in the right and left olfactory bulbs, and in the right and left olfactory cortices, respectively, showed similar values at the various survival intervals. Therefore, for the calculation of the results in Table 2 no dividing in right and left was done. It can be seen in Table 2 that the concentrations of 203Hg2` in the olfactory epithelium are considerable higher than in the brain at 1 day and 1 week. In the brain of these rats the 203Hg2` concentrations in the olfactory bulbs are statistically higher than in the olfactory cortex and the rest of the brain at all survival intervals.
Olfactory epithelium Olfactory bulbs Olfactory cortex Brainb Liver Kidney a b c
1 day
1 week
3 weeks
6 weeks
22$1.2
12$1.4
3.2$0.3
0.9$0.1
2.5$0.3 2.2$0.1b 1.8$0.3b 73$7.4 895$65
3.9$0.3 2.6$0.3b 2.9$0.2b 39$9.0 832$71
2.3$0.2 1.0$0.3b 1.3$0.1b 6.4$1.3 445$46
0.9$0.1 0.3$0.1b 0.4$0.1b 1.6$0.4 274$50
means$SD, n"5—6 rats. P40.05 compared to the olfactory bulbs (Wilcoxon signed rank test). Brain except olfactory bulb and olfactory cortex.
However, a comparison of the data in Table 2 with those in Table 1 shows that the high labeling of the right olfactory bulb is unique for the rats given the 203 Hg2` in the right nostril. At the 1- and 3-week survival intervals the levels of 203Hg2` in the olfactory cortex and the rest of the brain appear to be somewhat higher in the i.p. injected rats than in the i.n. injected ones, whereas at the 1-day and 6-week survival intervals similar values are seen in the i.p. and i.n. injected rats. A comparison of the data in Table 2 and Table 1 further shows that the concentrations of 203Hg2` in the kidney are higher after i.p. administration of the metal than after i.n. instillation at the corresponding survival intervals. Also the liver contains higher concentrations of the metal after i.p. compared to i.n. administration of the metal. Right—left ratios of 203Hg2`. It can be seen in Fig. 6 that in the rats given the metal in the right nostril the ratios between the right and left olfactory epithelium are higher than in the right and left olfactory bulb at 1 day. It can also be noted that the two rats which have the lowest ratios in the olfactory epithelium also have the lowest ratios in the olfactory bulbs. At the 1-, 3-, and 6-week survival intervals the ratios between the right and left olfactory epithelium and the right and left olfactory bulbs show similar values. In the olfactory cortex the ratios are close to 1 at all intervals after i.n. instillation of the metal. In the rats given 203Hg2` i.p. the ratios between the levels of the metal on the right and left side are close to 1 in the olfactory epithelium, the olfactory bulb, and the olfactory cortex at all survival intervals.
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FIG. 6. Ratios between the concentrations of 203Hg2` in the olfactory epithelium on the right and left side of the nasal septum and in the olfactory bulb and the olfactory cortex on the right and left side of the midline of the brain in rats given the metal i.n. or i.p. The rats were killed at different intervals after administration of the 203Hg2` (6.7 lg). The intranasal instillations were done in the right nostril. Each curve shows the data obtained in a single rat.
DISCUSSION
The demonstration that instillation of 203Hg2` in the right nostrils of the rats resulted in much higher levels of the metal in the right olfactory bulbs than in the left ones, together with the observation of absence of a similar uptake after i.p. administration, support the assumption that there is a direct passage of the metal from the olfactory epithelium to the olfactory bulbs. At the side of the application of the 203 Hg2` there was a labeling of the olfactory nerve bundles projecting to the olfactory bulbs, as well as in the olfactory neve-fibres constituting the olfactory nerve layer of the olfactory bulbs, which was not seen on the opposite side. The results also showed that the 203Hg2` accumulated in the glomerular layer of the bulbs. These data indicate that our observations can be ascribed to a movement of the mercury along the olfactory axons to their terminal parts in the glomeruli and not to circulatory uptake from the mucosal vasculature. The labeling was more selectively localized to the glomerular layer at 3 weeks than at 1 week. At 3 weeks there is a low source of mercury available for transport from the olfactory epithelium and most of the 203Hg2` appears
to have reached the axonal terminations in the glomeruli. At the 1- and 3-week survival intervals a weak labeling was also discernable in the external plexiform layer. This observation indicates that a low level of 203Hg2` leaves the terminal arborizations of the axons in the glomeruli. However, the labeling of the olfactory cortex, i.e., the projection field of the secondary olfactory neurons, did not differ from other ‘‘extrabulbar’’ brain areas. This indicates that the mercury which leaves the terminal parts of the primary olfactory neurons is not transferred via secondary olfactory neurons. Rather the mercury may diffuse in the extracellular space of the external plexiform layer. That there is a lack of transport of mercury via secondary olfactory neurons is further substantiated by the observation that the levels of the metal were similar in the right and left olfactory cortices of the rats given the 203Hg2` intranasally. In addition the levels of mercury in the olfactory cortex of the rats given the 203Hg2` i.p. did not differ very much from the levels in the i.n. injected rats and these concentrations were also similar to those observed in the rest of the brain. These data indicate that the mercury in the olfactory cortex and in the other extrabulbar areas is taken up from the blood.
UPTAKE OF INORGANIC MERCURY IN THE OLFACTORY BULBS
We have previously shown that 203Hg2` is taken up from the olfactory epithelium along the olfactory neurons to the olfactory bulbs of pikes (Borg-Neczak and Tja¨ lve, 1996). This transport occurs in an anterograde direction. Studies in rodents by Arvidson (1992) and Schi+nning (1993) have shown that inorganic mercury (Hg2`) is taken up in nerve terminals in skeletal muscles and then transported retrogradely along the motoneurons to the cell bodies in the spinal cord, the brainstem, and the dorsal root ganglia. Schi+nning (1993) reported that intraneuronal injection of colchicine, which is an inhibitor of cellular functions dependent on microtubules, suppressed the retrograde transport of mercury in the rat sciatic nerve, indicating an active intraneuronal movement of the metal. Further studies are required to determine the mechanism for the uptake of the mercury in the olfactory neurons. Following intranasal administration the mercury is probably taken up in the olfactory knobs, which constitute the terminal swellings of the dendritic processes, extending to the surface of the epithelium. It is known that the olfactory knobs are rich in endocytic vesicles, indicating that they are actively engaged in endocytosis of exogeneous materials from the extracellular environment (de Lorenzo, 1970; Bannister and Dodson, 1992). Entry of mercury by endocytotis may involve a binding of the metal to surface proteins followed by internalization. It is known that such a mechanism is involved in the uptake of various macromolecules in nerve terminals (Yamamoto et al., 1987). In addition Gutknecht (1981) suggested that Hg2` may pass biological membranes as an uncharged HgCl2 complex. The Hg2` may also enter the cells via sodium or calcium channels, as has been proposed as the mode of uptake of Hg2` in motor nerve terminals in frog muscle (Miyamoto, 1983). The autoradiography showed an uptake of 203Hg2` in the glomerular layer of the olfactory bulbs also after i.p. administration of the metal. This observation was supported by the gamma spectrometry, which showed significantly higher levels of mercury in the olfactory bulbs than in other parts of the brain. It can be noted that the gamma spectrometry reflects the average mercury concentration in the bulbs and not specificially the uptake in the glomerular layer. The results also showed an uptake of the mercury in the olfactory epithelium in the i.p. injected rats. We assume that the mercury is taken up from the blood into the primary olfactory neurons and then moves along the axons to their terminations in the bulb. Results by Arvidson (1992) have indicated a movement of mercury from peripheral
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nerve terminals along motoneurons to their cell bodies in the spinal cord and brainstem following intravenous injection of Hg2` in mice. In the rats injected i.p. with 203Hg2` we observed a labeling of the superior and inferior colliculi which slightly exceeded the background labeling of the brain. We also found a labeling of the optic nerves. The superior colliculus is a terminal of the axons of the retinal ganglion cells and conceivably the mercury may reach this area via transport in the optic nerves. The inferior colliculus is a terminal of the central auditory tract. Conceivably the mercury may reach this area via neurons in the auditory system. The habenula also showed a somewhat higher labeling than the background in the brain. The habenula receives fibers from the olfactory cortex via the olfactory—habenular tract (Allison, 1953), but it is not currently known whether mercury can be transported along this route. Our results showed a high accumulation of 203 Hg2` in plexus chorioideus in the rats given the metal i.p. It is known that inorganic mercury is accumulated in this structure (Berlin and Ullberg, 1963). As mentioned in the Introduction, there are indications that Hg0 is oxidized to Hg2` in the nasal mucosa (Khayat and Dencker, 1984). The oxidation is carried out by the H2O2-catalase pathway, which is ubiquitously present in various tissues (Sichak et al., 1986; Clarkson, 1989). It is well known that Hg0 is oxidized to Hg2` in the CNS. We propose that Hg0 released from amalgam fillings may reach the primary olfactory neurons in the olfactory epithelium and become oxidized to Hg2` in these cells. The intracellular Hg2` may then be transported along the axons. In humans a continuous exposure of the nasal mucosa to mercury released from amalgam fillings, as well as a potential uptake of the metal in the olfactory epithelium from the blood, may result in considerable concentrations of the metal in the olfactory bulbs. Maas and co-workers (1996) examined the concentration of mercury in the olfactory bulbs of 55 deceased persons and found significantly higher levels of the metal in the bulbs than in the occipital lobe cortex (geometric mean 17.4 lg/kg wet wt compared to 9.2 lg/kg wet wt). However, the levels of mercury in the olfactory bulbs could not be correlated to the number of amalgam fillings. It would be of interest to further explore the concentration of mercury in the olfactory bulbs in humans. The potential toxicity of mercury for the olfactory sense is also an issue which needs to be explored. To our knowledge this problem has not been examined
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in mammals. However, the olfactory system has been shown to be very vulnerable to mercury in fish (Hara et al., 1976; Baatrup et al., 1990). As mentioned Hg2` has been shown to be transported via the olfactory pathway in pike (Borg-Neczak and Tja¨ lve, 1996). It is also of interest to notice that the olfactory system has been shown to be impared in early stages of some neurodegenerative disorders, such as Alzheimer’s disease and Parkinson’s disease, and it has been proposed that an uptake of neurotoxic materials along the olfactory pathways may contribute to these diseases (Roberts, 1986; Ferrera-Moyano and Barragan, 1989; Doty, 1991). Therefore, toxicants which are able to enter the brain via the olfactory system deserve special attention. REFERENCES Allison, A. C. (1953). The morphology of the olfactory system in the vertebrates. Biol. Rev. 28, 195—244. Arvidson, B. (1992). Inorganic mercury is transported from muscular nerve terminals to spinal and brainstem motoneurons. Muscle Nerve 15, 1089—1094. Baatrup, E., D+ving, K. B., and Winberg, S. (1990). Differential effects of mercurial compounds on the electroolfactogram (EOG) of salmon (Salmo salar L.) Ecotoxicol. Environ. Saf. 20, 269—276. Berlin, M., and Ullberg, S. (1963). Accumulation and retention of mercury in the mouse. Arch. Environ. Health 6, 589—601. Bannister, L. H., and Dodson, H. C. (1992). Endocytic pathways in the olfactory and vomeronasal epithelia of the mouse: Ultrastructure and uptake of tracers. Microsc. Res. Tech. 23, 128—141. Borg-Neczak, K., and Tja¨ lve, H. (1996). Uptake of 203Hg2` in the olfactory system in pike. Toxicol. Lett. 84, 107—112. Clarkson, T. W. (1989). Mercury. J. Am. Coll. Toxicol. 8, 12911295. de Lorenzo, A. J. D. (1970). The olfactory neuron and the blood—brain barrier. In ‘‘Taste and Smell in Vertebrates’’ (G. E. W. Wolstenholme and J. Knight, Eds.), pp. 151—176. Churchill, London. Doty, R. L. (1991). Olfactory dysfunction in neurodegenerative disorders. In ‘‘Smell and Taste in Health and Disease’’ (T. V. Getchell, L. M. Bartoshuk, R. L. Doty, and J. B. Snow, Jr., Eds.), pp. 735—751. Raven Press, New York. Ferrera-Moyano, H., and Barragan, E. (1989). The olfactory system and Alzheimer’s disease. Int. J. Neurosci. 49, 157—197.
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